Abstract

The PhD project concerned the analysis of the primary and secondary structure of Glycosamminoglycans.
The aim of the work was the collection of new information (spectroscopic and conformational ones) about Hyaluronic acid, Chondroitin sulfate and Keratan sulfate, in order to amplify the literature data available about their structures-function relationship.
Glycosaminoglycans (GAGs), are a family of linear polydisperse polysaccharides that take part in several physiological phenomena, including neuronal development, cell–matrix interactions, and activation of chemokines, enzymes, and growth factors. These chains are typically linked to a protein core forming proteoglycans at the cell surface or in the extracellular matrix.
The ability of GAGs to regulate these processes is attributed to their complex structure, which arises from extensive modifications (sulfation, deacetylation and epimerization) of a nonsulfated precursor consisting of hexosamine (D-Glucosamine, D-Galactosamine) and either hexuronic acid (D-Glucuronic acid, L-iduronic acid) or galactose residues, arranged in an alternating linear sequence.
Based on of the chemical structure backbone, there are four classes of GAGs: Hyaluronan (HA), Heparin (HP)/Heparan sulfate (HS), Chondroitin (CS)/Dermatan sulfate (DS) and Keratan sulfate (KS).
Despite their simple backbone sequence, glycosaminoglycans are complex molecules characterized by a heterogeneous structure, for this reason the investigation on them is really challenging and it is performed usually combining many techniques.
In this work, analyses were carried out applying a combination of chemical, enzymatic and spectroscopic tools. In some cases the spectroscopic data collected were analyzed with the support of theoretical data obtained through molecular mechanics and dynamics simulations.
The complete work, for a easier organization, is divided into two parts:
•Structural characterization of natural GAG samples:
Chondroitin sulfate from the cartilage of Scyliorhinus canicula, Raja brachyura, and Torpedo nobiliana and also from the skin of Raja brachyura.
Keratan sulfate from the cartilage of the Scyliorhinus canicula.
•Conformational analyses of GAG oligosaccharides:
Decasaccharide of hyaluronic acid
Hexasaccharide of unsulfate chondroitin
Dodedecasaccharide of chondroitin sulfate
Pentasaccharides of keratan sulfate
Structural characterization of natural GAG samples
Structural analyses were performed on chondroitin sulfate and keratan sulfate chains isolated from natural source: cartilaginous fishes. Purification, quantification and separation of GAGs polysaccharides and their enzymatic-derived oligosaccharides were performed using different chromatographic approaches like anion exchange, size exclusion chromatography (SEC), strong anion exchange HPLC (SAX-HPLC) and reverse phase ion pairing HPLC (RPIP-HPLC).
Spectroscopic investigations on the intact molecules and on the different oligosaccharides isolated were performed with NMR spectroscopy.
Chondroitin sulfate from the cartilage of Scryliorhinus canicula, Raja brachyura, and Torpedo nobiliana and from the skin of Raja brachyura
Compositional analysis of all the CS molecules led to the identification of the same repeating units (CS-0S [4)-GlcA-(1→3)-GalNAc-(1-], CS-A [-4)-GlcA-(1→3)-GalNAc4S-(1-], CS-C [-4)-GlcA-(1→3)-GalNAc6S-(1-], and CS-D [-4)-GlcA2S-(1→3)-GalNAc6S-(1]), but occurring in different percentages. In the table below a summary of the results is reported:
Specimen CS0S CS-A CS-C CS-D
Scyliorhinus canicula
Cartilage 8.2% 41% 32% 19.8%
Raja bochyura
Cartilage 9.5% 36.5% 39.0% 15%
Raja bochyura
Skin 5.7% 37.1% 38.4% 18.8%
Torpedo nobiliana
Cartilage 0.9% 30.9% 38.2% 30%
This analysis, as expected, highlighted for all the samples a high negative charge density, and among the four CS samples, that isolated from the torpedo cartilage showed the higher charge density, since it was characterized by the higher content of disulfated motif and the minor content of the unsulfated one.
For the characterization of the CS isolated from the cartilage of Schilorhinus canicula a new quantitative spectroscopic method was applied (Q-HSQC) too and the data collected were confirmed by the classical analysis (SAX-HPLC) of the mixture of the unsaturated disaccharides obtained by enzymatic depolymerisation (ABC lyase digestion). This disaccharide mixture and that containing the linkage region glycopeptides, moreover, were separated by RPIP-HPLC leading to sample directly analyzed by 2D-NMR.
Characterization of the CS samples isolated from Raja Bochyura and Torpedo Nobiliana tissues was performed by a deep SAX-HPLC analysis of the oligosaccharides mixtures belonging to ABC and AC lyase depolymerisations.
The separation of the products resistant to AC lyase action has accidently led to the isolation of oligosaccharides with a structure different from the one expected, since peeling and degradation reactions occurred during the separation steps.
In this way besides the classical compositional analysis based on the SAX-HPLC separation of the unsaturated disaccharide mixture a collection of NMR data of CS tri and tetrasaccharides was achieved as well.
Keratan sulfate from the cartilage of the Scyliorhinus canicula:
Keratan sulfate material was classified as a O-linked glycan with a molecular weight around 10 kDa. The polysaccharide chain, as expected for shark KS, was characterized by GlcNAc6S units and both Gal and Gal6S units. The structure was not fucosilated, but resulted end-capped by sialic acid units.
Conformational analyses of GAG oligosaccharides
The conformational studies on Hyaluronic acid, Chondroitin sulfate and Keratan sulfate oligosaccharides were carried out through a combination of spectroscopic and molecular modelling techniques .
Decasaccharide of hyaluronic acid
The conformational behaviour of a HA decasaccharide was investigated through the analysis of theoretical data, collected by a molecular dynamics simulation in explicit water, with the support of spectroscopic data collected through Residual Dipolar Coupling NMR techniques.
This particular NMR technique was used because the anisotropic conditions (liquid-crystal medium) determine a partial alignment with respect to the external magnetic field and estimation of the relative orientation of the corresponding C-H or H-H vectors with respect to the magnetic field encodes for long range information.
The combined use of these NMR and molecular modelling techniques led to establish that in solution the HA tended to assume a quite elongate conformation well described by a left-handed 3-fold helix.
Hexasaccharide of unsulfate chondroitin
The secondary structure of unsulfated and sulfated CS oligosaccharides was investigated through the analysis of simulation data collected by MD simulations in explicit water as well, but the spectroscopic data used as reference were collected through the classic NMR spectroscopy in isotropic conditions: D2O or D2O:H2O mixture.
The conformational behaviour of the unsulfate CS molecule was similar to the one estimated for the HA, as expected on the basis of the structural similarity of the two chains, so, in this case a conformation compatible with a left-handed 3-fold helix geometry was found as well. Differently from HA, however, a higher flexibility for the -(1→3) linkage was estimated, as indicated by the larger fluctuation range found for the torsional angle  and by the weak H-bond network defined across the glycosidic junction.
Dodedecasaccharide of chondroitin sulfate
Investigation on a chondroitin sulfate model sequence, supported by the comparison with the data collected for the unsulfated molecule, highlighted that the presence of sulfate groups affected first of all the behaviour of the glycosidic torsional angles, and not the N-Acetyl disposition, the CH2OH preferred rotamer and the H-bond network , because they were similar to those detected for the unsulfated structure. In particular, the presence of sulfate groups led to a reduction of the fluctuation range of both phi and psi torsional angles, with a higher effect on psi one, that shifted in many towards negative values.
Pentasaccharides of keratan sulfate
Unsulfated and sulfated KS pentasaccharides were studied with molecular mechanics and dynamics simulations in implicit solvent. In this case, the effect of the presence of sulfate groups on the conformation of polylactosamine backbone was investigated. As found for the chondroitin sulfate case, both the glycosidic linkages were affected by the presence of these bulked and negative charged groups and a shift to positive psi values was detected, as well.